Biology subjects

Gupta, A. K.

Publications and source records attributed to Gupta, A. K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A Prodrug Strategy to Reposition Atovaquone as a Long-Acting Injectable for Malaria Chemoprotection

Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.

pharmacology and toxicology↗

bioRxiv2024

The mitochondrial thiolase ACAT1 regulates monocyte/macrophage type I interferon via epigenetic control.

Lipid-derived acetyl-CoA is shown to be the major carbon source for histone acetylation. However, there is no direct evidence demonstrating lipid metabolic pathway contribututions to this process. Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes the final step of {beta}-oxidation, the aerobic process catabolizing fatty acids (FA) into acetyl-CoA. To investigate this in the context of immunometabolism, we generated macrophage cell line lacking ACAT1. 13C-carbon tracing combined with mass spectrometry confirmed incorporation of FA-derived carbons into histone H3 and this incorporation was reduced in ACAT1 KO macrophage cells. RNA-seq identified a subset of genes downregulated in ACAT1 KO cells including STAT1/2 and interferon stimulated genes (ISGs). CHIP analysis demonstrated reduced acetyl-H3 binding to STAT1 promoter/enhancer regions. Increasing histone acetylation rescued STAT1/2 expression in ACAT1 KO cells. Concomitantly, ligand triggered IFN{beta} release was blunted in ACAT1 KO cells and rescued by reconstitution of ACAT1. Furthermore, ACAT1 promotes FA-mediated histone acetylation in an acetylcarnitine shuttle-dependent manner. In patients with obesity, levels of ACAT1 and histone acetylation are abnormally elevated. Thus, our study identified a novel link between ACAT1 mediated FA metabolism and epigenetic modification on STAT1/2 that uncovers a regulatory role of lipid metabolism in innate immune signaling and opens novel avenues for interventions in human diseases such as obesity.

immunology↗

bioRxiv2023

Discovery of CMX990: A Potent SARS-CoV-2 3CL Protease Inhibitor Bearing a Novel Covalent Warhead

There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof towards human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = PaxlovidTM), CMX990 has distinctly differentiated potency ([~]5x more potent in primary cells) and human in vitro clearance (>4x better microsomal clearance and >10x better hepatocyte clearance), with good in vitro-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.

microbiology↗

bioRxiv2023

Comparative Genomic and Transcriptomic Analysis of Avirulent and Virulent Strains of Fusarium oxysporum f. sp. carthami: Insights into Pathogenesis and Virulence Determinants in Safflower Infections

Vascular wilt disease incited by Fusarium oxysporum f. sp. carthami (Foc) in Safflower poses a significant threat to its production in India. A comprehensive understanding of the molecular underpinning of compatible and incompatible interaction is of extreme economic importance. In the present study, the genome of a virulent (IARI-5175) and a avirulent (F-00845) Foc strain were sequenced and assembled using data generated from Illumina in combination with Nanopore technologies and HiC. Foc genomes were assembled into 88 and 23 scaffolds with an estimated total size of 46 Mb and 42 Mb respectively for IARI-5175 and F-00845 strains. Reference based mapping of Foc genome with F. oxysporum f. sp. lycopersici (Fol) resulted in chromosomal level ordering of genome and simultaneous identification of accessory genome. Additionally, two lineage specific chromosomes were also identified for virulent Foc strain IARI-5175. Genomic comparisons were made on the basis of effectors, CAZymes, secondary metabolites and mycotoxins to understand the global view pathogenicity in Foc. Moreover, the transcriptome of Foc during compatible and incompatible interaction was sequenced and analyzed leading to the identification of differentially regulated genes. Taken together our study laid a solid foundation to explore novel effector genes that play a crucial role in the establishment of disease and can further be used as targets to devise new strategies to curb wilt disease in safflower.

genomics↗

bioRxiv2023

Transcription elongation defects link oncogenic splicing factor mutations to targetable alterations in chromatin landscape

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two splicing factors commonly mutated in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy. HIGHLIGHTS- Oncogenic mutations in SF3B1 and U2AF1 cause a gene body RNAPII transcription elongation defect - The elongation defect is linked to impaired assembly of early spliceosome complexes and leads to replication stress and changes to chromatin landscape - RNAPII elongation defects in SF3B1K700E are normalized by modulating epigenetic factors but 3 cryptic splicing events are not reversed - Targeting the Sin3/HDAC pathway to normalize RNAPII elongation defect is a potential therapeutic approach in these disorders GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/530019v3_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@dc6d22org.highwire.dtl.DTLVardef@180f36borg.highwire.dtl.DTLVardef@ed341forg.highwire.dtl.DTLVardef@f8dfb2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

biochemistry↗

bioRxiv2022

Combinatorial encoding of odors in the mosquito antennal lobe

Among the cues that a mosquito uses to find a host for blood-feeding, the smell of the host plays an important role. Previous studies have shown that host odors contain hundreds of chemical odorants, which are detected by different receptors on the peripheral sensory organs of mosquitoes. But how individual odorants are encoded by downstream neurons in the mosquito brain is not known. We developed an in vivo preparation for patch-clamp electrophysiology to record from projection neurons and local neurons in the antennal lobe of Aedes aegypti. Combining intracellular recordings with dye-fills, morphological reconstructions, and immunohistochemistry, we identify different sub-classes of antennal lobe neurons and their putative interactions. Our recordings show that an odorant can activate multiple neurons innervating different glomeruli, and that the stimulus identity and its behavioral preference are represented in the population activity of the projection neurons. Our results provide the first detailed description of olfactory neurons in the central nervous system of mosquitoes and lay a foundation for understanding the neural basis of their olfactory behaviors.

neuroscience↗

bioRxiv2022

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

microbiology↗

bioRxiv2022

Mosquito Olfactory Response Ensemble: a curated database of behavioral and electrophysiological responses enables pattern discovery

Many experimental studies have examined behavioral and electrophysiological responses of mosquitoes to odors. However, the differences across studies in data collection, processing, and reporting make it difficult to perform large-scale analyses combining data from multiple studies. Here we extract and standardize data for 12 mosquito species, along with Drosophila melanogaster for comparison, from over 170 studies and curate the Mosquito Olfactory Response Ensemble (MORE), publicly available at https://neuralsystems.github.io/MORE. We demonstrate the ability of MORE in generating biological insights by finding patterns across studies. Our analyses reveal that ORs are tuned to specific ranges of several physicochemical properties of odorants; the empty-neuron recording technique for measuring OR responses is more sensitive than the Xenopus oocyte technique; there are systematic differences in the behavioral preferences reported by different types of assays; and odorants tend to become less attractive or more aversive at higher concentrations.

neuroscience↗

bioRxiv2021

Functional Network Connectivity Imprint In Febrile Seizures

Complex febrile seizures (CFS), a subset of paediatric febrile seizures (FS), have been studied for their prognosis, epileptogenic potential and neurocognitive outcome. We evaluated their functional connectivity differences with simple febrile seizures (SFS) in children with recent-onset FS. Resting-state fMRI (rs-fMRI) datasets of 24 children with recently diagnosed FS (SFS-n=11; CFS-n=13) were analysed. Functional connectivity (FC) was estimated using time series correlation of seed region-to-whole-brain-voxels and network topology was assessed using graph theory measures. Regional connectivity differences were correlated with clinical characteristics (FDR corrected p < 0.05). CFS patients demonstrated increased FC of the bilateral middle temporal pole (MTP), and bilateral thalami when compared to SFS. Network topology study revealed increased clustering coefficient and decreased participation coefficient in basal ganglia and thalamus suggesting an inefficient-unbalanced network topology in patients with CFS. The number of seizure recurrences negatively correlated with the integration of Left Thalamus (r= -0.58) and FC measures of Left MTP to Right Supplementary Motor and left Precentral connectivity (r=-0.53). The FC of Right MTP to Left Amygdala, Putamen, Parahippocampal, and Orbital Frontal Cortex (r=0.61) and FC of Left Thalamus to left Putamen, Pallidum, Caudate, Thalamus Hippocampus and Insula (r 0.55) showed a positive correlation to the duration of the longest seizure. The findings of the current study report altered connectivity in children with CFS proportional to the seizure recurrence and duration. Regardless of the causal/consequential nature, such observations demonstrate the imprint of these disease-defining variables of febrile seizures on the developing brain.

neuroscience↗

bioRxiv2021

Large-scale mRNA translation and the intricate effects of competition for the finite pool of ribosomes

We present a new theoretical framework for large-scale mRNA translation using a network of models called the ribosome flow model with Langmuir kinetics (RFMLK), interconnected via a pool of free ribosomes. The input to each RFMLK depends on the pool density, and it affects the initiation rate and the internal ribosome entry rates at each site along each RFMLK. Ribosomes that detach from an RFMLK due to termination or premature drop-off are fed back into the pool. We prove that the network always converges to a steady-state, and study its sensitivity to variations in the parameters. For example, we show that if the drop-off rate at some site in some RFMLK is increased then the pool density increases and consequently the steady-state production rate in all the other RFMLKs increases. Surprisingly, we also show that modifying a parameter of a certain RFMLK can lead to arbitrary effects on the densities along the modified RFMLK, depending on the parameters in the entire network. We conclude that the competition for shared resources generates an indirect and intricate web of mutual effects between the mRNA molecules, that must be accounted for in any analysis of translation.

systems biology↗

bioRxiv2021

Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2)

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3 untranslated regions (3UTRs) of mRNA, but the identity of functional targets remains unknown. Here we used iCLIP to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of target mRNAs largely in 3UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more "U/TAG" motifs compared to targets bound non-productively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute MSI2 loss. Eukaryotic elongation factor 3A (EIF3A) was determined to be an immediate, direct target. We propose that MSI2 down-regulation of EIF3A amplifies these effects on the proteome. Our results also underscore the challenges in defining functional targets of RBP since mere binding does not imply a discernible functional interaction.

cancer biology↗

bioRxiv2020

EVALUATION OF ANTI-BACTERIAL AND ANTHELMINTIC ACTIVITIES OF BARK OF PLANT CASSIA FISTULA Linn.

This study deals with phytochemical screening and evaluation of anti-bacterial and anthelmintic activities of the bark extracts of Cassia fistula L. The extraction with different solvents - methanol, acetone, distilled water - had been carried out. The anti-bacterial assay was done against two gram +ve and two gram -ve bacteria by agar cup diffusion method. The anthelmintic activity was done on Pheretima posthuma (earthworm) by recording the paralyzing and death time under different concentrations. Methanolic extract showed the maximum activity on both fronts be it anti-bacterial or anthelmintic. The extract was effective only against gram +ve bacteria. Among Staphylococcus aureus and Streptococci faecalis, the former was more susceptible. The activity against gram -ve bacteria was not found. In case of anthelmintic activity, the concentration of 50 mg/ml was effective and near to the paralyzing and death time as recorded for the 10 mg/ml concentration of the standard Albendazole. However, aqueous extract was more effective than the acetone extract.

pharmacology and toxicology↗

bioRxiv2020

Refinement of Genome assemblies of Pigeonpea (Cajanus cajan)

Genome assembly of short reads from large plant genomes remains a challenge in computational biology despite major developments in Next Generation sequencing. Of late multiple draft assemblies of plant genomes are reported in many organisms. The draft assemblies of Cajanus cajan are with different levels of genome completeness; contain large number of repeats, gaps and segmental duplications. Draft assemblies with portions of genome missing, are shorter than the referenced original genome. These assemblies come with low map accuracy affecting further functional annotation and prediction of gene component as desired by crop researchers. Genome coverage i.e. number of sequenced raw reads mapped on to certain locations of the genome is an important quality indicator of completeness and assembly quality in draft assemblies. Present work was aimed at improvement of coverage in reported de novo sequenced draft genomes (GCA_000340665.1 and GCA_000230855.2) of Pigeonpea, a legume widely cultivated in India. The two assemblies comprised 72% and 75% of estimated coverage of genome respectively. We employed assembly reconciliation approach to compare draft assemblies and merged them to generate a high quality near complete assembly with enhanced contiguity. Finished assembly has reduced number of gaps than reported in draft assemblies and improved genome coverage of 82.4%. Quality of the finished assembly was evaluated using various quality metrics and for presence of specific trait related functional genes. Employed pair-end and mate-pair local library data sets enabled to resolve gaps, repeats and other sequence errors yielding lengthier scaffolds compared to two draft assemblies. We report prediction of putative host resistance genes from improved sequence against Fusarium wilt disease and evaluated them in both wet laboratory and field phenotypic conditions.

genomics↗

bioRxiv2020

Oral drug repositioning candidates and synergistic remdesivir combinations for the prophylaxis and treatment of COVID-19

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. A high-throughput, high-content imaging assay of human HeLa cells expressing the SARS-CoV-2 receptor ACE2 was used to screen ReFRAME, a best-in-class drug repurposing library. From nearly 12,000 compounds, we identified 66 compounds capable of selectively inhibiting SARS-CoV-2 replication in human cells. Twenty-four of these drugs show additive activity in combination with the RNA-dependent RNA polymerase inhibitor remdesivir and may afford increased in vivo efficacy. We also identified synergistic interaction of the nucleoside analog riboprine and a folate antagonist 10-deazaaminopterin with remdesivir. Overall, seven clinically approved drugs (halofantrine, amiodarone, nelfinavir, simeprevir, manidipine, ozanimod, osimertinib) and 19 compounds in other stages of development may have the potential to be repurposed as SARS-CoV-2 oral therapeutics based on their potency, pharmacokinetic and human safety profiles.

microbiology↗

bioRxiv2020

Abundance and diversity of phages, microbial taxa and antibiotic resistance genes in the sediments of the river Ganges through metagenomic approach

In the present study, we have analyzed the metagenomic DNA from the pooled sediment sample of the river Ganges to explore the abundance and diversity of phages, microbial community and antibiotic resistance genes. Utilizing data from Illumina platform, 4174 ([~]0.0013%) reads were classified for the 285 different DNA viruses largely dominated by the group of 260 distinctive phages (3602 reads, [~]86.3%). Among all, Microcystis (782 hits), Haemophilus (403), Synechococcus (386), Pseudomonas (279), Enterococcus (232), Bacillus (196), Rhodococcus (166), Caulobacter (163), Salmonella (146), Enterobacteria (143), Mycobacterium (128), Propionibacterium (71), Erwinia (70), Ralstonia (56) phages shows the highest abundance and account for approximately 90% of the total identified phages. Additionally, we have also identified corresponding host pertaining to these phages. Mainly, Proteobacteria ([~]69.3%) dominates the microbial population structure. Primarily orders such as Caulobacterales ([~]28%), Burkholderiales ([~]13.9%), Actinomycetales ([~]13.7%), Pseudomonadales ([~]7.5%) signify the core section. Further, 21869 ([~]0.00695%) reads were classified in 20 ARG types (classes) and 240 ARGs (subtypes) among which 4 ARG types namely multidrug resistance (MDR) (12041 reads, [~]55%), bacitracin (3202 reads, [~]15%), macrolide-lincosamide-streptogramin (MLS) (1744 reads, [~]7.98%), and fosmidomycin (990 reads, [~]4.53%) has the highest abundance. Simultaneously, six resistance mechanisms were also recognized with the dominance of antibiotic efflux (72.8%, 15919 reads). The results unveil the distribution of (pro)-phages; microbial community and various ARGs in the Ganges river sediments. Further research on these identified phage(s) could be used in phage-based therapeutics against pathogenic bacteria.

genomics↗

bioRxiv2019

Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids

Recent years have seen rapid advances in directed differentiation of human pluripotent stem cells (PSCs) to kidney cells. However, a fundamental difficulty in emulating kidney tissue formation is that kidney development is iterative. Recent studies argue that the human nephron forms through gradual contribution of nephron progenitor cells whose differentiation fates depend on the time at which they are recruited. We show that the majority of PSC-derived nephron progenitor cells differentiated in a short wave in organoid formation and to improve fidelity of PSC-derived organoids, we emulated the asynchronous mix found in the fetal kidney by combining cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and lineage marking data showed that proximal and distal nephron components preferentially derive from cell populations differentiated at distinct times. When engrafted under the kidney capsule these heterochronic organoids were vascularized and displayed essential features of kidney tissue. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed using intravenous injection of fluorescent dextran. Despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study, and we propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop the same stromal overgrowth when engrafted under the kidney capsule.

developmental biology↗

bioRxiv2018

Integrated ligand and structure-based investigation of structural requirements for silent information regulator activation

A series of imidazothiazole and oxazolopyridine derivatives as human silent information regulator (SIRT1) activators were subjected to the integrated 2D and 3D QSAR approaches. The derived 3D QSAR models yielded high cross validated q2 values of 0.682 and 0.628 for CoMFA and CoMSIA respectively. The non-cross validated correlation values of r2training = 0.89; predictive r2test = 0.69 for CoMFA and r2=0.87; predictive r2test =0.67 for CoMSIA reflected the statistical significance of the developed model. The steric, electrostatic, hydrophobic and hydrogen bond acceptor interactions have been found important in describing the variation in human SIRT1 activation. Further, 2D QSAR model for the same dataset yielded high statistical significance and derived 2D models parameters corroborated with 3D model in terms of features. The developed model was also validated through the available active conformation structure of SIRT1. Developed models may be useful for the identification of potential novel human SIRT1 activators as therapeutic agent.

biophysics↗